Process for the Production of beta-Amino Acids Using Acylase

ABSTRACT

The present invention relates to a  Variovorax  sp. which produces an acylase having an asymmetric hydrolysis activity on an N-acetyl β-amino acid to selectively produce an R-β-amino acid, and a  Burkholderia  sp. which produces both an acylase having an asymmetric hydrolysis activity on an N-acetyl β-amino acid to selectively produce an S-β-amino acid and an acylase having an asymmetric hydrolysis activity of an N-acetyl β-amino acid to selectively produce an R-β-amino acid, and a process for the selective production of an S-, or R-β-amino acid using the above strains.

This application is a divisional application under 35 U.S.C. §120 toU.S. patent application Ser. No. 11/198,242, filed Aug. 8, 2005, whichclaimed priority under 35 U.S.C. §119 to Japanese Patent Application No.2004-231306, filed Aug. 6, 2004, the contents of both of which areincorporated by reference in their entireties. The Sequence Listingfiled electronically herewith is also hereby incorporated by referencein its entirety (File Name: US-245D; File Size: 5 KB; Date Created: Oct.26, 2007).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the selective productionof an S-β-amino acid or an R-β-amino acid by reacting a racemic compoundof N-acetyl β-amino acid with an acylase having an asymmetric hydrolysisactivity on the N-acetyl β-amino acid, and to novel microorganisms thatmay be used in the above process.

2. Background Art

β-amino acids are non-naturally occurring amino acids that are notcomponents of animal proteins. Like other non-naturally occurring aminoacids, the β-amino acids have been in demand more as intermediatecompounds for medical products, and have been used as ingredients insome medical products (Chimica OGGI/Chemistry today 10, 15 (2002)).

The following three methods are already known for production of β-aminoacids using acylase:

(1) Method with Penicillin G Acylase (Formula 1)

This method is most popular and is referred to in, for example, V. A.Soloshonok et al., Tetrahedron, 6, 1601 (1995) and the like. This methoduses phenylacetylation of the amino group of a racemic compound such as3-amino-3-phenylpropionic acid (referred to hereinafter as “β-phenylalanine”), followed by division using Penicillin G acylase toselectively produce its R-form.

(2) Method with Porcine Kidney Acylase I (Formula 2)

The amino group of the racemic compound of β-phenylalanine ischloroacetylated and N-chloroacetyl-β-phenylalanine is divided usingporcine kidney acylase I to selectively produce its S-form(WO2003/080854A2, published 02.10.1003, Degussa Co., or Grayson & K.Drauz CHIMICA OGGI/chemistry today 10, 5 (2002)). According to thespecification, the reaction did not substantially occur whenN-acetyl-β-phenylalanine was used.

(3) Method with Glutaryl 7-aminocephalosporanic Adid Acylase (Formula 3)

The amino group of the racemic compound of β-phenylalanine isglutarylated and N-gluaryl-β-phenylalanine is divided using Glutaryl7-aminocephalosporanic acid acylase to selectively produce its R-form(WO2003/020943A2, published 13. 03. 2003, Aventis Pharma).

SUMMARY OF THE INVENTION

For normal α-amino acids, the amino group is generally acetylatedfollowed by division using S- or R-selective acylase. However, nohydrolase has been described for N-acetyl-β-amino acids.

Acetylation is most commonly used since the N-acetyl group is readilyformed by merely reacting an amino acid with acetic anhydride. However,as mentioned above, phenylacetylation, chloroacetylation, andglutarylation have been used for β-amino acids, since there is noacylase for hydrolyzing the N-acetyl group.

For solving the above problems, the present inventors have studiedintensively and found novel microorganisms that produce an enzymecapable of selectively hydrolyzing the N-acetyl group to its S-form orR-form. The present inventors have further confirmed that the S-form orR-form of β-amino acids may be freely produced using thesemicroorganisms, and have accordingly completed the present invention.

It is an object of the present invention to provide a Variovorax sp.strain which produces an acylase having an asymmetric hydrolysisactivity on an N-acetyl β-amino acid so that an R-β-amino acid isselectively produced.

It is a further object of the present invention to provide theVariovorax sp. strain described above wherein the N-acetyl β-amino acidcomprises N-acetyl β-phenylalanine.

It is an object of the present invention to provide the Strain 119L (AJ110348, FERM ABP-10367).

It is an object of the present invention to provide a Burkholderia sp.strain which produces both an acylase having an asymmetric hydrolysisactivity on an N-acetyl β-amino acid to selectively produce an S-β-aminoacid, and an acylase having an asymmetric hydrolysis activity on anN-acetyl β-amino acid to selectively produce R-β-amino acid.

It is an object of the present invention to provide the Burkholderia sp.strain according to claim 4 wherein the N-acetyl β-amino acid comprisesN-acetyl β-phenylalanine.

It is a further object of the present invention to provide the Strain130F (AJ 110349, FERM ABP-10366).

It is an object of the present invention to provide a process for theselective production of an S-β-amino acid or an R-β-amino acidcomprising reacting a racemic compound of N-acetyl β-amino acid with anacylase having an asymmetric hydrolysis activity on said N-acetylβ-amino acid.

It is an object of the present invention to provide a process for theselective production of an R-β-amino acid as described above comprisingreacting a racemic compound of N-acetyl β-amino acid with an acylasehaving an asymmetric hydrolysis activity on the N-acetyl β-amino acid.

It is an object of the present invention to provide a process for theselective production of an S-β-amino acid by the strain as describedabove comprising reacting at a range of about 25˜about 30° C. a racemiccompound of N-acetyl β-amino acid with the acylase having an asymmetrichydrolysis activity on the N-acetyl β-amino acid.

A process for the selective production of an R-β-amino acid by thestrain as described above comprising reacting at a range of about75˜about 80° C. a racemic compound of N-acetyl β-amino acid with anacylase having an asymmetric hydrolysis activity on the N-acetyl β-aminoacid.

The process as described above, wherein the racemic compound of N-acetylβ-amino acid is added into a suspension of the strain so to react withthe acylase.

The process as described above, wherein the racemic compound of N-acetylβ-amino acid is added into a supernatant of the strain breakage solutionso to react with the acylase.

Thus, the present invention provides strains which produce an acylasehaving an asymmetric hydrolysis activity on an N-acetyl β-amino acid toselectively produce an S-, or R-β-amino acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photos of the electrophoresis, which show the production ofβ-Phe from N-acetyl-DL-β-Phe (Left: 119L; Right: 130F). 1: DL-β-Phe 0.5%solution, N-acetyl-DL-β-Phe 0.5% solution, 3: Reaction solution withoutthe enzyme, 4: Time 0, 5: Time 3 hours.

FIG. 2 shows photos of the electrophoresis, which shows the substratespecificity of β-Phe aminoacylase (Left: 119L; Right: 130F). 1: DL-β-Phe0.5% solution, N-acetyl-DL-β-Phe 0.5% solution, 3: Before reaction, 4:Reaction solution of D-form, 5: D-form at time 0, 6: D-form at time 3hours, 7: Reaction solution of L-form, 8: L-form at time 0, 9: L-form attime 3 hours.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Variovorax sp. of the present invention is characterized by producing anacylase having an asymmetric hydrolysis activity on an N-acetyl-β-aminoacid to selectively produce an R-β-amino acid.

Burkholderia sp. of the present invention is characterized by producingboth an acylase having an asymmetric hydrolysis activity on an N-acetylβ-amino acid to selectively produce an S-β-amino acid and an acylasehaving an asymmetric hydrolysis activity on an N-acetyl β-amino acid toselectively produce an R-β-amino acid. As demonstrated in the followingexamples, the optimal reaction temperature of an acylase to selectivelyproduce the S-form ranges between about 25° C. and about 30° C.,preferably around 30° C., and the optimal reaction temperature of anacylase to selectively produce the R-form ranges between about 75° C.and about 80° C., preferably around 75° C.

There is no limitation on the N-acetyl β-amino acid, which can berepresented by the following formula 4:

R1 can represent a C1-C6 alkyl group, a C6-C10 aryl group, a C7-C11aralkyl group, or the same groups containing a hetero atom, which mayhave a substituent. R2 represents an hydrogen atom or hydroxy group.N-acetyl β-phenylalanine and its derivatives are preferred.

A strain that belongs to the Variovorax sp. of the present inventionincludes the strain 119L (AJ 110348), which was deposited on Jul. 22,2004 at the International Patent Organism Depository, National Instituteof Advanced Industrial Science and Technology and granted the numberFERM AP-20129. This deposit was converted to a deposit under theBudapest Treaty on Jul. 4, 2005 and granted number FERM ABP-10367.

A strain that belongs to the Burkholderia sp. of the present inventionincludes the strain 130F (AJ 110349), which was deposited on Jul. 22,2004 at the International Patent Organism Depository, National Instituteof Advanced Industrial Science and Technology and granted the numberFERM AP-20128. This deposit was converted to a deposit under theBudapest Treaty on Jul. 4, 2005 and granted number FERM ABP-10366.

Their mycological features are described in detail in the examples.

The reaction using the Variovorax sp. according to the present inventionmakes it possible to selectively hydrolyze the R-form of N-acetylβ-amino acid to produce R-β-amino acid while leaving N-acetyl(S)-β-aminoacid.

The reaction using Burkholderia sp according to the present invention,for example, at a temperature of 30° C. makes it possible to selectivelyhydrolyze the S-form of N-acetyl β-amino acid to produce S-β-amino acidwhile leaving N-acetyl(R)-β-amino acid. On the other hand, the reactionwith the same strain, for example, at a temperature of 75° C. makes itpossible to selectively hydrolyze the R-form of N-acetyl β-amino acid toproduce R-β-amino acid while leaving N-acetyl(S)-β-amino acid. Aceticacid is generated as a by-product.

The reaction scheme using N-acetyl-β-phenylananine as the N-acetylβ-amino acid is shown in formula 5 below.

The present invention relates therefore to a process for the selectiveproduction of an S-β-amino acid or an R-β-amino acid by reacting aracemic compound of N-acetyl β-amino acid with an acylase having anasymmetric hydrolysis activity on the N-acetyl β-amino acids.

There is no limitation on the origin or preparation method of theacylase that is used in the above process, as long as it has the desiredactivity. Therefore, the acylase may be produced by the above strains,and prepared from the same strains by any method known to those skilledin the art. Alternatively, the enzyme produced by a recombinant hostcell transformed by the gene encoding the acylase may also be utilized.

The manners, methods, conditions, and the like of the above reactionwith the acylase in the present process may be optionally selected fromthose known to those skilled in the art depending on various conditions,such as the kind and amount of the acylase to be used, and productionscale.

For example, the racemic compound of N-acetyl β-amino acid may be addedinto the suspension of the strain or into the supernatant of the strainbreakage solution so to react with the acylase.

The present invention will be explained more in detail with reference tothe following non-limiting examples.

Example 1 Chemical synthesis of N-acetyl-DL-3-amino-3-phenylpropionicacid (Ac-β-Phe)

β-Phe (45.3 g, manufactured by Aldrich) was mixed with 182 ml of water.Then, 32 ml of ice-cooled 25% sodium hydroxide aqueous solution wasadded to this mixture. The resulting solution was adjusted to pH 11.4.Acetic anhydride (58 ml) was dropped while being adjusted to pH 11˜12with 25% sodium hydroxide aqueous solution. The reaction solution wasthen heated to 40° C. and stirred for 3.5 hours while being adjusted topH 11˜12. After the reaction was complete, the insoluble matters wereremoved by filtration, and the resulting filtrate was ice-cooled again.The solution was adjusted to pH 2 by adding 105 ml of conc. HCl followedby crystallization for 2 hours. The precipitated crystal was separatedand washed with 100 ml of water. It was dried under reduced pressure at40° C. to give a yield of 94% white crystals of Ac-β-Phe (53.4 g)

TLC analysis was carried out in the following examples by development ona silica gel 60F254 (Merck) with a developing solvent (butanol/aceticacid/water=4/1/2), and detection of the products by coloring withninhydrin or absorbance at 254 nm. N-acetyl-DL-β-Phe prepared as abovewas used as the standard, N-acetyl-L-β-Phe (N-acetyl-(R)-β-Phe: preparedfrom L-β-Phe ((R)-β-Phe) manufactured by Watanabe Chemical Industry Inc.by the similar method), N-acetyl-D-β-Phe (N-acetyl-(S)-β-Phe: preparedfrom D-β-Phe ((S)-β-Phe) manufactured by Watanabe by the similarmethod), DL-β-Phe (Aldrich), L-β-Phe ((R)-β-Phe: Watanabe ChemicalIndustry Inc.), D-β-Phe ((RS)-β-Phe: Watanabe Chemical Industry Inc.).

Example 2 Searching for β-Phe Acylase

A microorganism producing an enzyme that can hydrolyze the acetyl groupof Ac-β-Phe with an optical specificity was screened in the followingmanner.

Screening of a microorganism having β-Phe aminoacylase:

Soil samples (200 samples) collected at various Kanto areas in Japanwere inoculated into a synthetic culture medium (ammonium sulfate 10.0g/l, KH₂PO₄ 1.0 g/l, MgSO₄.7H₂O 0.4 g/l, FeSO₄.7H₂O 10 mg/l, MnSO₄.5H₂O10 mg/, vitamin B1.HCl 0.2 mg/l, yeast extract 0.5 μl, N-acetyl-DL-β-Phe5.0 g/l, pH8.0) containing chemically synthesized N-acetyl derivative ofDL-β-Phe, and cultured with shaking. The enzyme reaction was done in theculture broth that showed growth of the microorganism.

The enzyme reaction was conducted by mixing one part 0.2 M phosphatebuffer solution (pH 6.5) containing 1.0% N-acetyl-DL-β-Phe and one partthe suspension of the strain or the supernatant of the strain breakagesolution, and reacting for 3 hours at 31.5° C. After the reaction wasterminated by incubating it for 5 min at 90° C., the reaction solutionwas analyzed by TLC analysis.

As a result, generation of β-Phe from N-acetyl derivative of DL-β-Phewas found in 4 samples. Microorganisms were isolated from the culturebroth of these 4 samples, and a hydrolyzing activity of the DL-β-Phederivatives was examined for the isolated strains. The microorganismshaving β-Phe aminoacylase activity were successfully isolated from twostrains (119L, 130F).

The mycological classification of the two strains was done by total basesequence analysis of 16SrDNA, and a homology search was conducted basedon the total base sequence analysis (MicroSeq Microbial Full Libraryv.0001 (Applied Biosystems, CA, USA)), and molecular phylogenetic treeby proximate connection method (MicroSeq Microbial IdentificationSystgeneem Software V.1.4.1), which were consigned to NCIB Japan. Incase any strain showing 100% identity in the homology search was notfound, the classification was done by homology search using DNA basesequence data base (GenBank/DDBJ/EMBL) with BLAST. The total 16SrDNAsequences of the strains 119L and 130F are shown in Table 1, as Seq. IDN0.1, and in Table 2, as Seq. ID N0.2, respectively. Furthermore, theirmycological features (morphological characters and physiologicalcharacters) are summarized in Table 3 and Table 4 (the strain 119L) andTable 5 and Table 6 (the strain 130F).

TABLE 1    1 gagtttgatc ctggctcaga ttgaacgctg gcggcatgcc ttacacatgc   51aagtcgaacg gcagcgcggg agcaatcctg gcggcgagtg gcgaacgggt  101 gagtaatacatcggaacgtg cccaatcgtg ggggataacg cagcgaaagc  151 tgtgctaata ccgcatacgatctacggatg aaagcagggg atcgcaagac  201 cttgcgcgaa tggagcggcc gatggcagattaggtagttg gtgaggtaaa  251 ggctcaccaa gccttcgatc tgtagctggt ctgagaggacgaccagccac  301 actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 351 ttttggacaa tgggcgcaag cctgatccag ccatgccgcg tgcaggatga  401aggccttcgg gttgtaaact gcttttgtac ggaacgaaac ggctctttct  451 aataaagagggctaatgacg gtaccgtaag aataagcacc ggctaactac  501 gtgccagcag ccgcggtaatacgtagggtg caagcgttaa tcggaattac  551 tgggcgtaaa gcgtgcgcag gcggtgatgtaagacagttg tgaaatcccc  601 gggctcaacc tgggaactgc atctgtgact gcatcgctggagtacggcag  651 agggggatgg aattccgcgt gtagcagtga aatgcgtaga tatgcggagg 701 aacaccgatg gcgaaggcaa tcccctgggc ctgtactgac gctcatgcac  751gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgcccta  801 aacgatgtcaactggttgtt gggtcttcac tgactcagta acgaagctaa  851 cgcgtgaagt tgaccgcctggggagtacgg ccgcaaggtt gaaactcaaa  901 ggaattgacg gggacccgca caagcggtggatgatgtggt ttaattcgat  951 gcaacgcgaa aaaccttacc cacctttgac atgtacggaatttgccagag 1001 atggcttagt gctcgaaaga gaaccgtaac acaggtgctg catggctgtc1051 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1101cttgtcatta gttgctacat tcagttgggc actctaatga gactgccggt 1151 gacaaaccggaggaaggtgg ggatgacgtc aagtcctcat ggcccttata 1201 ggtggggcta cacacgtcatacaatggctg gtacaaaggg ttgccaaccc 1251 gcgaggggga gctaatccca taaaaccagtcgtagtccgg atcgcagtct 1301 gcaactcgac tgcgtgaagt cggaatcgct agtaatcgtggatcagaatg 1351 tcacggtgaa tacgttcccg ggtcttgtac acaccgcccg tcacaccatg1401 ggagcgggtt ctgccagaag tagttagctt aaccgcaagg agggcgatta 1451ccacggcagg gttcgtgact ggggtgaagt cgtaacaagg tagccgtatc 1501 ggaaggtgcggctggatcac ctcctt

TABLE 2    1 gagtttgatc ctggctcaga ttgaacgctg gcggcatgcc ttacacatgc   51aagtcgaacg gcagcgcggg ggcaaccctg gcggcgagtg gcgaacgggt  101 gagtaatacatcggaacgtg tcctgtagtg ggggatagcc cggcgaaagc  151 cggattaata ccgcatacgctctacggagg aaaggggggg atcttaggac  201 ctctcgctac aggggcggcc gatggcagattagctagttg gtggggtaaa  251 ggcctaccaa ggcgacgatc tgtagctggt ctgagaggacgaccagccac  301 actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 351 ttttggacaa tgggcgcaag cctgatccag caatgccgcg tgtgtgaaga  401aggccttcgg gttgtaaagc acttttgtcc ggaaagaaaa cgccgtggtt  451 aatacccgtggcggatgacg gtaccggaag aataagcacc ggctaactac  501 gtgccagcag ccgcggtaatacgtagggtg caagcgttaa tcggaattac  551 tgggcgtaaa gcgtgcgcag gcggtccgctaagacagatg tgaaatcccc  601 gggcttaacc tgggaactgc atttgtgact ggcgggctagagtatggcag  651 aggggggtag aattccacgt gtagcagtga aatgcgtaga gatgtggagg 701 aataccgatg gcgaaggcag ccccctgggc caatactgac gctcatgcac  751gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgcccta  801 aacgatgtcaactagttgtt ggggattcat ttccttagta acgtagctaa  851 cgcgtgaagt tgaccgcctggggagtacgg tcgcaagatt aaaactcaaa  901 ggaattgacg gggacccgca caagcggtggatgatgtgga ttaattcgat  951 gcaacgcgaa aaaccttacc tacccttgac atgtatggaatcctgctgag 1001 aggtgggagt gcccgaaagg gagccataac acaggtgctg catggctgtc1051 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1101cttgtcccta gttgctacgc aagagcactc tagggagact gccggtgaca 1151 aaccggaggaaggtggggat gacgtcaagt cctcatggcc cttatgggta 1201 gggcttcaca cgtcatacaatggtcggaac agagggtcgc caacccgcga 1251 gggggagcca atcccagaaa accgatcgtagtccggatcg cactctgcaa 1301 ctcgagtgcg tgaagctgga atcgctagta atcgcggatcagcatgccgc 1351 ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac accatgggag1401 tgggttttac cagaagtggc tagtctaacc gcaaggagga cggtcaccac 1451ggtaggattc atgactgggg tgaagtcgta acaaggtagc cgtatcggaa 1501 ggtgcggctggatcacctcc tt

TABLE 3 1. Morphological characteristics: Culture conditions: Nutrientagar (Oxoid, Hampshire, England) medium, 30° C. Form of cell: 0.6-0.7 ×1.5-2.0 μm Polymorphism: − Mobility (the state + of adherence offlagella): Spore (site of spores): − 2. Culture characteristics: Cultureconditions: Nutrient agar (Oxoid, Hampshire, England) medium, 30° C.Color: Yellow Luster + Production of pigment: + Culture conditions:Nutrient agar (Oxoid, Hampshire, England) medium, 30° C. The presence ofsurface growth: + Turbidity of media: + Culture conditions: Gelatin stabculture, 30° C. State of growth: − Gelatin liquefaction¹⁾: − Cultureconditions²⁾: Litmus milk, 30° C. Coagulation: − Liquefaction: − 3.Physiological characteristics: Gram stain¹⁾ − Reduction of nitrate³⁾ −Denitrification²⁾ − MR test²⁾ − VP test³⁾ + Production of indole³⁾ −Production of hydrogen sulfide³⁾ − Hydrolysis of starch²⁾ − Utilizationof citric acid²⁾ (Koser) + Utilization of citric acid²⁾ − (Christensen)Utilization of inorganic + nitrogen²⁾ (nitrate) Utilization ofinorganic + nitrogen²⁾ (ammonium salt) Catalase²⁾ + Oxidase²⁾ + Range ofgrowth (pH 5): + Range of growth (pH 8): + Range of growth (pH10): −Range of temperature (20° C.): + Range of temperature (25° C.): + Rangeof temperature (37° C.): +w Range of temperature (45° C.): − Behaviortoward anaerobic: + O-F test (Oxidation/Fermentation)²⁾: −/−

TABLE 4 4. Formation of acids/gases²⁾ L-arabinose −/− D-xylose −/−D-glucose −/− D-mannose −/− D-fluctose −/− D-galactose −/− Maltose −/−Sucrose −/− Lactose −/− Trehalose −/− D-sorbiotl −/− D-mannitol −/−Inositol −/− Glycerin −/− 5. Other physiological characteristics:³⁾β-Galactosidase activity: − Arginine dihydrolase activity: − Lysinedecarboxylase activity: − Tryptophan deaminase activity: − Gelatinaseactivity: − References and Kit 1) BARROW, (G. I.) and FELTHAM, (R. K.A.): Cowan and Steel's Manual for the Identification of MedicalBacteria. 3rd edition, 1993, Cambridge University Press. ²⁾ToshikazuSakazaki et al., “New Lectures on Bacterial Culture, “2nd edition, 1988,Kindai Publisher, Tokyo ³⁾Kit for identification of bacteria API20 NE(bioMerieux, France: http://www.biomerieux.fr/home_en.htm)

TABLE 5 1. Morphological characteristics: Culture conditions: Designatedmedium, 30° C. Form of cell: 0.5-0.6 × 1.0-1.5 μm Polymorphism: −Mobility (the state of + adherence of flagella): Spore (site of spores):− 2. Culture characteristics: Culture conditions: Designated medium, 30°C. Color: Cream Luster + Production of pigment: − Culture conditions:Nutrient agar (Oxoid, Hampshire, England) medium, 30° C. The presence ofsurface growth: − Turbidity of media: + Culture conditions: Gelatin stabculture, 30° C. State of growth: − Gelatin liquefaction¹⁾: − Cultureconditions²⁾: Litmus milk, 30° C. Coagulation: − Liquefaction: − 3.Physiological characteristics: Gram stain¹⁾ − Reduction of nitrate³⁾ +Denitrification²⁾ − MR test²⁾ − VP test³⁾ + Production of indole³⁾ −Production of hydrogen sulfide³⁾ − Hydrolysis of starch²⁾ − Utilizationof citric acid²⁾ + (Koser) Utilization of citric acid²⁾ + (Christensen)Utilization of inorganic nitrogen²⁾ + (nitrate) Utilization of inorganicnitrogen²⁾ + (ammonium salt) Urease activity³⁾ − Catalase²⁾ +Oxidase²⁾ + Range of growth (pH 5): + Range of growth (pH 8): +w Rangeof growth (pH10): − Range of temperature (20° C.): +w Range oftemperature (25° C.): + Range of temperature (37° C.): + Range oftemperature (45° C.): − Behavior toward anaerobic: − O-F test(Oxidation/Fermentation)²⁾: +/−

TABLE 6 4. Formation of acids/gases²⁾ L-arabinose +/− D-xylose +/−D-glucose −/− D-mannose −/− D-fluctose +/− D-galactose −/− Maltose +/−Sucrose −/− Lactose −/− Trehalose +/− D-sorbiotl −/− D-mannitol −/−Inositol −/− Glycerin −/− 5. Other physiological characteristics:³⁾β-Galactosidase activity: + Arginine dihydrolase activity: − Lysinedecarboxylase activity: − Tryptophan deaminase activity: − Gelatinaseactivity: − References and Kit 4) BARROW, (G. I.) and FELTHAM, (R. K.A.): Cowan and Steel's Manual for the Identification of MedicalBacteria. 3rd edition, 1993, Cambridge University Press. 5) ToshikazuSakazaki et al., “New Lectures on Bacterial Culture, “2nd edition, 1988,Kindai Publisher, Tokyo 6) Kit for identification of bacteria API20 NE(bioMerieux, France: http://www.biomerieux.fr/home_en.htm)

Example 3 Determination of β-Phe Aminoacylase Activity

Variovorax sp. (FERM ABP-10367) or Burkholderia sp. (FERM ABP-10366) wasinoculated into 20 ml of a culture medium (1% (NH₄)₂SO₄, 0.1% KH₂PO₄,0.04% MgSO₄.7H₂O, 10 ppm FeSO₄.7H₂O, 10 ppm MnSO₄.7H₂O, 0.2 ppm VitaminB1 hydrochloride, 1% Glucose, 0.1% NaCl, 0.1% MES, 0.2% casamino acid,0.5% N-acetyl-DL-β-Phe, pH7.0) in a shaking flask (500 ml) and culturedwith shaking at 135 rpm and 31.5° C. for 24 or 48 hours. The strainswere collected from the culture broth by centrifugation (8,000 ppm×15min). A part of the collected strains was suspended in 0.2 M phosphatebuffer (pH 6.5) and disrupted by ultrasonic treatment (300 W, 3.5 min,4° C.). The disrupted strains were subjected to centrifugation (8,000ppm×15 min) to remove the precipitate, giving supernatant of the strainbreakage solution.

The enzyme reaction and TLC analysis as described above using the thusobtained strains and the supernatant of the strain breakage solutionconfirmed the enzyme activity in the strains and the supernatant. Theseresults suggested that the enzymes were intracellular. As these enzymeswere produced only when the strains were cultured in a medium containingthe substrate, N-acetyl-DL-β-Phe, they were considered to be aninducible enzyme. The results are shown in FIG. 1.

Example 4 Examination of the Substrate-Specificity of β-Phe Aminoacylase

The same enzyme reaction and TLC analysis were carried out usingN-acetyl-L-β-Phe (N-acetyl-(R)-β-Phe) and N-acetyl-D-β-Phe(N-acetyl-(S)-β-Phe) instead of N-acetyl-DL-β-Phe (FIG. 2).

The results showed that Variovorax sp. (FERM ABP-10367) selectivelyhydrolyzed N-acetyl-L-β-Phe (N-acetyl-(R)-β-Phe) to produce only L-β-Phe((R)-β-Phe) without any production of S-β-Phe, as seen from Table 7. Allthe data shown in Table 7 or herein after were determined by HPLC.

TABLE 7 Time Concentration (mM) (min) N—Ac—R-β-Phe N—Ac—S-β-Phe R-β-PheS-β-Phe 0 0.63 0.63 0.00 0.00 1 0.62 0.63 0.01 0.00 5 0.39 0.64 0.170.00 10 0.20 0.64 0.37 0.00 15 0.08 0.64 0.50 0.00 20 0.02 0.64 0.580.00 25 0.00 0.63 0.59 0.00 30 0.00 0.64 0.60 0.00

This data shows that the optimal pH range is between pH 7.5 and 8.0, asshown in Table 8. The results regarding the optimal reaction temperatureare shown in Table 9 (reaction time: 10 min).

TABLE 8 R-β-Phe Production Relative R—N—Ac-β-Phe(mM) Ratio (mM) ActivitypH 0 min 10 min (%) 100 mM Na Acetate-HCl 3.5 0.63 0.00 0.00 4.0 0.500.00 0.00 5 0.57 0.00 0.00 5.5 0.56 0.00 0.00 100 mM MES-NaOH 5.5 0.620.00 0.00 6.0 0.48 0.05 26.3 6.5 0.44 0.08 40.2 7.0 0.32 0.13 61.5 100mM Tris-HCl 7.0 0.35 0.14 71.0 7.5 0.29 0.20 100.00 8.0 0.29 0.20 96.09.0 0.42 0.07 34.3

TABLE 9 Reaction Temperature (° C.) R-β-Phe (mM) Relative Activity (%)25 0.22 48.6 30 0.31 70.5 35 0.37 83.9 40 0.41 92.3 45 0.44 100 50 0.4295.6 55 0.26 59.4 60 0.16 36.5 65 0.12 27.8 70 0.00 0.0 75 0.00 0.0

It was found that Burkholderia sp. (FERM ABP-10366) selectivelyhydrolyzed N-acetyl-D-β-Phe (N-acetyl-(S)-β-Phe) at 30° C. to produceonly D-β-Phe ((S)-β-Phe), as seen from Table 10. The same reaction wasthen carried out for 15 min varying the reaction temperature, giving theresults shown in Table 11.

TABLE 10 Time Concentration (mM) (min)) N—Ac—R-β-Phe N—Ac—S-β-PheR-β-Phe S-β-Phe 0 0.60 0.62 0.00 0.00 1 0.60 0.57 0.00 0.03 2 0.61 0.540.00 0.05 5 0.60 0.52 0.00 0.10 7 0.61 0.44 0.00 0.12 10 0.57 0.39 0.000.15 12 0.57 0.33 0.00 0.15 15 0.56 0.31 0.00 0.19 17 0.58 0.30 0.000.21 20 0.59 0.28 0.00 0.24 25 0.58 0.20 0.00 0.29 30 0.57 0.14 0.000.34 35 0.56 0.10 0.02 0.41 40 0.56 0.06 0.00 0.42 50 0.57 0.00 0.020.42

TABLE 11 Reaction Temperature Concentration (mM) (° C.) N—Ac—R-β-PheN—Ac—S-β-Phe R-β-Phe S-β-Phe 25 0.54 0.20 0.00 0.16 30 0.58 0.21 0.000.28 35 0.57 0.00 0.10 0.38 40 0.59 0.00 0.09 0.42 45 0.31 0.01 0.300.39 50 0.31 0.10 0.33 0.35 55 0.00 0.08 0.52 0.33 60 0.00 0.20 0.670.31 65 0.00 0.29 0.47 0.23 70 0.15 0.36 0.44 0.13 75 0.46 0.55 0.070.00 80 0.57 0.56 0.00 0.00

As seen from the results in Table 11, while S-β-Phe was selectivelyproduced at the reaction temperature of 30° C. or below, R-β-Phe wasselectively produced at the reaction temperature of 75° C. or above.

Example 5 Reaction with Derivatives of β-Phenylalanine

The derivatives of β-phenylalanine and corresponding N-acetylderivatives were prepared as follows, and the same experiments were doneas above.

Synthesis of p-bromo-β-phenylalanine

p-Bromo-benzaldehyde (10.0 g, 54 mmol) was mixed with 100 ml ofethanol/water (95/5), 8.3 g of ammonium acetate (108 mmol), and 11.2 gof malonic acid (108 mmol), heated under reflux at 80° C. with stirringfor 17 hours, filtered under heating and dried under reduced pressure togive 8.3 g of p-bromo-β-phenylalanine crystals at a yield of 62.8%.

Synthesis of acetyl-p-bromo-β-phenylalanine

p-Bromo-β-phenylalanine (10.0 g, 41 mmol) was mixed with 40 ml of waterand adjusted to pH 14 with 25% sodium hydroxide aqueous solution. Afterbeing cooled to 5° C., 8.5 ml of acetic anhydride (90 mmol) and 25%sodium hydroxide aqueous solution were simultaneously dropped into themixture to keep it at a pH range of 11.5˜12 and the resulting mixturewas stirred for 3 hours at a room temperature. After the mixture wasadjusted to pH 2 with conc. HCl and stirred for one hour, the resultingprecipitated crystal was filtered and dried under reduced pressure togive quantitatively 12.0 g of acetyl-p-bromo-β-phenylalanine.

Synthesis of p-nitro-β-phenylalanine

Acetic acid (110 ml) was mixed with 20.4 g of ammonium acetate (264mmol) and heated to 85° C. After ammonium acetate was dissolved, themixture was further mixed with 20.0 g of p-nitro-benzaldehyde (132 mmol)and 27.8 g of malonic acid (264 mmol) and heated at 90° C. with stirringfor 5 hours. After being cooled to room temperature, the filtrate wasmixed with 300 ml of 2-propanol. The precipitated crystal was filtered,subjected to slurry washing with 100 ml of ethanol, filtered again anddried under reduced pressure to give 14.4 g of p-nitro-β-phenylalanineat a yield of 52.0%.

Synthesis of acetyl-p-nitro-β-phenylalanine

p-Nitro-β-phenylalanine (10.0 g, 48 mmol) was mixed with 40 ml of waterand adjusted to pH 14 with 25% sodium hydroxide aqueous solution. Afterbeing cooled to 5° C., 10.7 ml of acetic anhydride (105 mmol) and 25%sodium hydroxide aqueous solution were simultaneously dropped into themixture to keep it at a pH range of 11.5˜12 and the resulting mixturewas stirred for 1.5 hours at a room temperature. After the mixture wasadjusted to pH 2 with conc. HCl and stirred for one hour, the resultingprecipitated crystal was filtered and dried under reduced pressure togive 11.6 g of acetyl-p-nitro-β-phenylalanine at a yield of 96.9%.

Synthesis of 3,4-(—O—CH₂—O—)-β-phenylalanine

Piperonal (7.5 g, 50 mmol) was mixed with 75 ml of ethanol and 8.0 g ofammonium acetate (100 mmol), stirred at 40° C. and dissolved therein. Tothis solution 10.5 g of malonic acid (100 mmol) was added, and heatedfor 5 hours under reflux. After ethanol was concentrated under reducedpressure, the mixture was mixed with 75 ml of water and conc. HCl toadjust to pH2 and stirred for one hour. After the resulting precipitatedcrystal was filtered out, the filtrate was adjusted with 25% sodiumhydroxide aqueous solution to pH6, mixed with 75 ml of methanol andstirred overnight. The resulting precipitated crystal was filtered,subjected to slurry washing with 25 ml of 2-propanol, filtered again,and dried under reduced pressure to give 4.6 g of3,4-(—O—CH₂—O—)-β-phenylalanine at a yield of 43.5%.

Synthesis of acetyl-3,4-(—O—CH₂—O—)-β-phenylalanine

3,4-(—O—CH₂—O—)-β-phenylalanine (1.4 g, 7 mmol) was mixed with 28 ml ofwater and adjusted to pH 12 with 25% sodium hydroxide aqueous solution.After being cooled to 5° C., 1.6 ml of acetic anhydride (17 mmol) and25% sodium hydroxide aqueous solution were simultaneously dropped intothe mixture to keep it at a pH range of 11.5˜12 and the resultingmixture was stirred for 2.5 hours at 40° C. After the mixture wasfiltered, the filtrate was adjusted to pH 2 with conc. HCl and stirredovernight. The resulting precipitated crystal was filtered and driedunder reduced pressure to give 1.6 g ofacetyl-1-3,4-(—O—CH₂—O—)-β-phenylalanine at a yield of 94.0%.

The reaction of N-acetyl compounds thus synthesized in the same way withVariovorax sp. (FERM ABP-10367) and Burkholderia sp. (FERM ABP-10366) asin Example 2 revealed that the same selective hydrolysis occurred.

Since N-acetyl compounds of β-amino acids may be used as a startingmaterial in the process for the selective production of the S-β-aminoacid or the R-β-amino acid in the present invention, this process has anadvantage in view of cost compared to the conventional processes.Furthermore, as acetic acid generated as a by-product in the presentprocess is a safe substance, it is also very environmentally favorable.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changed can be made, and equivalents employed, withoutdeparting from the scope of the invention. All documents cited hereinare hereby incorporated by reference.

1. A process for the selective production of an R-β-amino acid by anisolated Variovorax sp. strain comprising a 16SrDNA sequence of SEQ IDNO: 1, comprising reacting a racemic compound of N-acetyl β-amino acidwith an acylase comprising an asymmetric hydrolysis activity on theN-acetyl β-amino acid.
 2. A process for the selective production of anS-β-amino acid by an isolated Burkholderia sp. strain comprising a16SrDNA sequence of SEQ ID NO: 2, comprising reacting at about 25° C. toabout 30° C. a racemic compound of N-acetyl β-amino acid with an acylasecomprising an asymmetric hydrolysis activity on the N-acetyl β-aminoacid.
 3. A process for the selective production of an R-β-amino acid byan isolated Burkholderia sp. strain comprising a 16SrDNA sequence of SEQID NO: 2, comprising reacting at about 75° C. to about 80° C. a racemiccompound of N-acetyl β-amino acid with an acylase comprising anasymmetric hydrolysis activity on the N-acetyl β-amino acid.
 4. Aprocess of claim 1 wherein said racemic compound of N-acetyl β-aminoacid is reacted with the acylase by adding it to a suspension of thestrain.
 5. A process of claim 1 wherein the racemic compound of N-acetylβ-amino acid is added into the supernatant of the strain breakagesolution so to react with the acylase.
 6. The process of claim 2 whereinthe racemic compound of N-acetyl β-amino acid is added into suspensionof the strain so to react with the acylase.
 7. The process of claim 2wherein the racemic compound of N-acetyl β-amino acid is reacted withthe acylase by adding it to the supernatant of a strain breakagesolution.
 8. The process of claim 3 wherein the racemic compound ofN-acetyl β-amino acid is reacted with the acylase by adding it to asuspension of the strain.
 9. The process of claim 3 wherein the racemiccompound of N-acetyl β-amino acid is reacted with the acylase by addingit to the supernatant of a strain breakage solution.